MST1 promotes apoptosis through regulating Sirt1-dependent p53 deacetylation

Mammalian Sterile 20-like kinase 1 (MST1) protein kinase plays an important role in the apoptosis induced by a variety of stresses. The MST1 is a serine/threonine kinase that is activated upon apoptotic stimulation, which in turn activates its downstream targets, JNK/p38, histone H2B and FOXO. It has been reported that overexpression of MST1 initiates apoptosis by activating p53. However, the molecular mechanisms underlying MST1-p53 signaling during apoptosis are unclear. Here, we report that MST1 promotes genotoxic agent-induced apoptosis in a p53-dependent manner. We found that MST1 increases p53 acetylation and transactivation by inhibiting the deacetylation of Sirtuin 1 (Sirt1) and its interaction with p53 and that Sirt1 can be phosphorylated by MST1 leading to the inhibition of Sirt1 activity. Collectively, these findings define a novel regulatory mechanism involving the phosphorylation of Sirt1 by MST1 kinase which leads to p53 activation, with implications for our understanding of signaling mechanisms during DNA damage-induced apoptosis.     

Notch1 Signaling Sensitizes Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis in Human Hepatocellular Carcinoma Cells by Inhibiting Akt/Hdm2-mediated p53 Degradation and Up-regulating p53-dependent DR5 Expression

Notch signaling plays a critical role in regulating cell proliferation, differentiation, and apoptosis. Our previous study showed that overexpression of Notch1 could inhibit human hepatocellular carcinoma (HCC) cell growth by arresting the cell cycle and inducing apoptosis. HCC cells are resistant to apoptotic induction by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), so new therapeutic approaches have been explored to sensitize HCC cells to TRAIL-induced apoptosis. We are wondering whether and how Notch1 signaling can enhance the sensitivity of HCC cells to TRAIL-induced apoptosis. In this study, we found that overexpression of ICN, the constitutive activated form of Notch1, up-regulated p53 protein expression in HCC cells by inhibiting proteasome degradation. p53 up-regulation was further observed in human primary hepatocellular carcinoma cells after activation of Notch signaling. Inhibition of the Akt/Hdm2 pathway by Notch1 signaling was responsible for the suppression of p53 proteasomal degradation, thus contributing to the Notch1 signaling-mediated up-regulation of p53 expression. Accordingly, Notch1 signaling could make HCC cells more sensitive to TRAIL-induced apoptosis, whereas Notch1 signaling lost the synergistic promotion of TRAIL-induced apoptosis in p53-silenced HepG2 HCC cells and p53-defective Hep3B HCC cells. The data suggest that enhancement of TRAIL-induced apoptosis by Notch1 signaling is dependent upon p53 up-regulation. Furthermore, Notch1 signaling could enhance DR5 expression in a p53-dependent manner. Taken together, Notch1 signaling sensitizes TRAIL-induced apoptosis in HCC cells by inhibiting Akt/Hdm2-mediated p53 degradation and up-regulating p53-dependent DR5 expression. Thus, our results suggest that activation of Notch1 signaling may be a promising approach to improve the therapeutic efficacy of TRAIL-resistant HCC.Notch signaling determines cell fate and affects cell proliferation, differentiation, and apoptosis during cell development (1). As a highly conserved family, Notch coordinates a signaling cascade present in all animal species studied to date (2). Mammals have four Notch receptors that bind five different ligands, among which Notch1 signaling functions in many physiological and pathophysiological processes of numerous cell types, and its dysfunction results in a variety of developmental defects, including embryonic lethality and adult disorders. For example, the Notch1/Jagged1 signaling pathway is activated during liver regeneration and is potentially contributing to signals affecting hepatocyte growth (3, 4). Inducible inactivation of Notch1 has been shown to cause nodular regenerative hyperplasia in mouse liver (5). These studies suggest that Notch1 signaling may be involved in the liver functions and the pathogenesis of liver diseases. Our previous study demonstrated that Notch1 signaling could suppress the growth of human hepatocellular carcinoma (HCC)⁴ cells by arresting the cell cycle and inducing apoptosis (6). However, the underlying molecular mechanisms remain to be fully understood.p53, an important tumor suppressor gene, is involved in cell cycle arrest and cellular apoptosis. Its activity is mostly regulated by complex networks of post-translational modifications, including phosphorylation, ubiquitination, and proteasome degradation. One protein that is essential for determining p53 stability is Mdm2 (mouse double minute protein 2) (7). Mdm2, a nuclear phosphoprotein and an E3 ubiquitin ligase, binds to p53 and ubiquitinates p53, leading to proteosome degradation of p53 (8). Another important mechanism of p53 stability is related to its phosphorylation status, which is Mdm2-dependent or Mdm2-independent (9). As to the regulation of p53 by Notch1, there are controversial reports that Notch1 activation increased p53 expression in neural progenitor cells (10); however, suppression of p53 by Notch signaling was also well established in lymphomagenesis (11). We also reported that Notch1 signaling significantly up-regulated p53 expression in SMMC7721 HCC cells (6); however, the molecular mechanisms remained unclear and needed to be further characterized.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of a superfamily of cell death-inducing ligands, induces apoptosis in a broad range of transformed cells and tumor cells but has little or no effect on normal cells (12). Therefore, TRAIL has been regarded as a potential drug for cancer therapy (12, 13). However, several kinds of cancer, including HCC, are not sensitive to soluble TRAIL treatment (14). HCC accounts for 80–90% of liver cancers and is one of the most prevalent carcinomas throughout the world, especially in Africa and Asia. Thus, it is worthwhile to find a new strategy to overcome the resistance of HCC cells to TRAIL-induced apoptosis.Considering that Notch1 signaling up-regulates p53 and induces apoptosis of HCC cells and that there are no reports to date that address the relationship between Notch1 signaling and TRAIL-induced apoptosis, in this study, we investigated whether and how Notch1 signaling could sensitize HCC cells to TRAIL-induced apoptosis. We demonstrate that Notch1 signaling up-regulates p53 expression by inhibiting proteasome degradation via, at least in part, suppressing the phosphatidylinositol 3-kinase/Akt/Hdm2 pathway. In addition, we here report that Notch1 signaling enhances DR5 (death receptor 5) expression in a p53-dependent manner, and DR5 contributes, at least in part, to the enhancement of TRAIL-induced apoptosis by Notch1 signaling. Accordingly, Notch1 signaling sensitizes HCC cells to TRAIL-induced apoptosis.     

LncRNA MST1P2/miR‐133b axis affects the chemoresistance of bladder cancer to cisplatin‐based therapy via Sirt1/p53 signaling

Although bladder cancer is commonly chemosensitive to standard first‐line therapy, the acquisition of the resistance to cisplatin (DDP)‐based therapeutic regimens remains a huge challenge. Noncoding RNAs (ncRNAs), including long noncoding RNAs (lncRNAs) and microRNAs, have been reported to play a critical role in cancer resistance to DDP. Here, we attempted to provide a novel mechanism by which the resistance of bladder cancer to DDP treatment could be modulated from the perspective of ncRNA regulation. We demonstrated that lncRNA MST1P2 (lnc‐MST1P2) expression was dramatically upregulated, whereas miR‐133b expression was downregulated in DDP‐resistant bladder cancer cell lines, SW 780/DDP and RT4/DDP. Lnc‐MST1P2 and miR‐133b negatively regulated each other via targeting miR‐133b. Both lnc‐MST1P2 silence and miR‐133b overexpression could resensitize DDP‐resistant bladder cancer cells to DDP treatment. More important, miR‐133b could directly target the Sirt1 3′‐untranslated region to inhibit its expression. Inc‐MST1P2/miR‐133b axis affected the resistance of bladder cancer cells to DDP via Sirt1/p53 signaling. In conclusion, MST1P2 serves as a competing endogenous RNA for miR‐133b to counteract miR‐133b‐induced suppression on Sirt1, therefore enhancing the resistance of bladder cancer cells to DDP. MST1P2/miR‐133b axis affects the resistance of bladder cancer cells to DDP via downstream Sirt1/p53 signaling.     

过表达UHRF2通过上调p53及p21表达引起HEK293细胞G1期阻滞

UHRF2（ubiquitin like with PHD and ring finger domains 2）是新近发现的具有多个结构域的核蛋白,在细胞周期调控和表观遗传学中发挥重要作用．近期研究提示,UHRF2是肿瘤抑制蛋白p53的1个E3连接酶,在体内外能与p53相互结合并促进其泛素化,过表达UHRF2能使细胞周期停滞于G1期．然而,UHRF2介导的G1期阻滞及其与p53联系尚不清楚．通过共转染UHRF2质粒及p53特异性小干扰RNA(siRNAs)到HEK293细胞构建细胞模型,探索UHRF2引起细胞周期停滞与p53之间的关系．结果显示,UHRF2能促进HEK293细胞中p53的稳定,从而引起p21 (CIP1/WAF1)基因表达,并使细胞周期停滞于G1期;但在siRNA抑制p53的表达后p21(CIP1/WAF1)表达降低,UHRF2引起的细胞周期阻滞消除．研究结果提示,UHRF2可通过稳定p53,上调p21的表达,从而介导细胞周期阻滞于G1期;同时UHRF2可能参与细胞周期调控及DNA损伤反应（DNA damage response, DDR）．UHRF2稳定p53的具体分子机制及其在DDR中的作用有待进一步研究证明．     

Oxysterol-binding Protein-related Protein 8 (ORP8) Increases Sensitivity of Hepatocellular Carcinoma Cells to Fas-Mediated Apoptosis

Human hepatoma (HCC) has been reported to be strongly resistant to Fas-mediated apoptosis. However, the underlying mechanisms are poorly understood. In this study the function of oxysterol-binding protein-related protein 8 (ORP8) in human hepatoma cells apoptosis was assessed. We found that ORP8 is down-regulated, whereas miR-143, which controls ORP8 expression, is up-regulated in clinical HCC tissues as compared with liver tissue from healthy subjects. ORP8 overexpression triggered apoptosis in primary HCC cells and cell lines, which coincided with a relocation of cytoplasmic Fas to the cell plasma membrane and FasL up-regulation. Co-culture of HepG2 cells or primary HCC cells with Jurkat T-cells or T-cells, respectively, provided further evidence that ORP8 increases HCC cell sensitivity to Fas-mediated apoptosis. ORP8-induced Fas translocation is p53-dependent, and FasL was induced upon ORP8 overexpression via the endoplasmic reticulum stress response. Moreover, ORP8 overexpression and miR-143 inhibition markedly inhibited tumor growth in a HepG2 cell xenograft model. These results indicate that ORP8 induces HCC cell apoptosis through the Fas/FasL pathway. The role of ORP8 in Fas translocation to the plasma membrane and its down-regulation by miR-143 offer a putative mechanistic explanation for HCC resistance to apoptosis. ORP8 may be a potential target for HCC therapy.     

Oxidative Stress-induced Inhibition of Sirt1 by Caveolin-1 Promotes p53-dependent Premature Senescence and Stimulates the Secretion of Interleukin 6 (IL-6)

Oxidative stress can induce premature cellular senescence. Senescent cells secrete various growth factors and cytokines, such as IL-6, that can signal to the tumor microenvironment and promote cancer cell growth. Sirtuin 1 (Sirt1) is a class III histone deacetylase that regulates a variety of physiological processes, including senescence. We found that caveolin-1, a structural protein component of caveolar membranes, is a direct binding partner of Sirt1, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82–101) to the caveolin-binding domain of Sirt1 (amino acids 310–317). Our data show that oxidative stress promotes the sequestration of Sirt1 into caveolar membranes and the interaction of Sirt1 with caveolin-1, which lead to inhibition of Sirt1 activity. Reactive oxygen species stimulation promotes acetylation of p53 and premature senescence in wild-type but not caveolin-1 null mouse embryonic fibroblasts (MEFs). Either down-regulation of Sirt1 expression or re-expression of caveolin-1 in caveolin-1 null MEFs restores reactive oxygen species-induced acetylation of p53 and premature senescence. In addition, overexpression of caveolin-1 induces stress induced premature senescence in p53 wild-type but not p53 knockout MEFs. Phosphorylation of caveolin-1 on tyrosine 14 promotes the sequestration of Sirt1 into caveolar membranes and activates p53/senescence signaling. We also identified IL-6 as a caveolin-1-specific cytokine that is secreted by senescent fibroblasts following the caveolin-1-mediated inhibition of Sirt1. The caveolin-1-mediated secretion of IL-6 by senescent fibroblasts stimulates the growth of cancer cells. Therefore, by inhibiting Sirt1, caveolin-1 links free radicals to the activation of the p53/senescence pathway and the protumorigenic properties of IL-6.     

Effects of Angiotensin II Type 2 Receptor Overexpression on the Growth of Hepatocellular Carcinoma Cells In Vitro and In Vivo

Increasing evidence suggests that the renin-angiotensin system (RAS) plays an important role in tumorigenesis. The interaction between Angiotensin II (AngII) and angiotensin type 1 receptor (AT1R) may have a pivotal role in hepatocellular carcinoma (HCC) and therefore, AT1R blocker and angiotensin I-converting enzyme (ACE) inhibitors may have therapeutic potential in the treatment of hepatic cancer. Although the involvement of AT1R has been well explored, the role of the angiotensin II Type 2 receptor (AT2R) in HCC progression remains poorly understood. Thus, the aim of this study was to explore the effects of AT2R overexpression on HCC cells in vitro and in mouse models of human HCC. An AT2R recombinant adenoviral vector (Ad-G-AT2R-EGFP) was transduced into HCC cell lines and orthotopic tumor grafts. The results indicate that the high dose of Ad-G-AT2R-EGFP–induced overexpression of AT2R in transduced HCC cell lines produced apoptosis. AT2R overexpression in SMMC7721 cells inhibited cell proliferation with a significant reduction of S-phase cells and an enrichment of G1-phase cells through changing expression of CDK4 and cyclinD1. The data also indicate that overexpression of AT2R led to apoptosis via cell death signaling pathway that is dependent on activation of p38 MAPK, pJNK, caspase-8 and caspase-3 and inactivation of pp42/44 MAPK (Erk1/2). Finally, we demonstrated that moderately increasing AT2R expression could increase the growth of HCC tumors and the proliferation of HCC cells in vivo. Our findings suggest that AT2R overexpression regulates proliferation of hepatocellular carcinoma cells in vitro and in vivo, and the precise mechanisms of this phenomenon are yet to be fully determined.     

Thr 446 phosphorylation of PKR by HCV core protein deregulates G2/M phase in HCC cells

Hepatitis C virus (HCV) is the major causative viral agent of cirrhosis and hepatocarcinoma (HCC). HCV core protein affects cell homeostasis, playing an important role in viral pathogenesis of HCC. We investigate the effects of HCV core protein expression on cell growth in HCC cell lines. Cell cycle distribution analysis of HepG2 polyclonal core positive cells reveals a peculiar accumulation of cells in G2/M phase. Different pathways mediate G2/M arrest: such as p53 and double strand RNA protein kinase (PKR). Flow cytometry in p53-null cells demonstrates that p53 plays only a marginal role in inducing HCV core-dependent G2/M phase accumulation that seems to be significantly affected by the functional inactivation of PKR. HCC core positive cells are characterized by a significant PKR phosphorylation in Thr 446 residue, which leads deregulation of mitosis. Moreover, we observe that the overexpression of the viral protein induces an upregulation of PKR activity, which does not correlate with an increased eIF-2 phosphorylation. This uncommon behavior of PKR suggests that its activation by HCV core protein could involve alternative PKR-dependent pathways, implicated in core-dependent G2/M accumulation. The described biological effects of HCV core protein on cell cycle could be an additional viral mechanism for both HCV resistance to interferon (IFN) and HCC HCV-related pathogenesis.     

p53 Promotes proteasome-dependent degradation of oncogenic protein HBx by transcription of MDM2

Hepatitis B virus X protein (HBx) is closely involved in the development of hepatocellular carcinoma (HCC). Tumor suppressor p53 was reported to induce HBx degradation and repress its oncogenic function recently, but the molecular mechanism is unknown. In this study, we attempted to identify the underlying mechanism. We found that overexpression of p53 protein reduces the level of HBx protein and shortens its half-life, however, in MDM2 knock out cells, p53 has no effects on degradation of HBx, meanwhile, overexpression of MDM2 in absence of p53 can accelerate turnover of HBx protein. These indicate that p53-mediated HBx degradation is MDM2-dependent. MDM2 interacts with HBx in vitro and in vivo but does not promote its ubiquitination. In consistent with the results above, HCC tissue samples with wild-type p53 hardly detect HBx protein, whereas, HBx always accumulate in the tissues with mutant p53. Our data provide a possible mechanism on how p53 regulate HBx stability and also a new clue for the study of p53 mutation and HCC development.     

Targeted p21(WAF1/CIP1) Activation by RNAa Inhibits Hepatocellular Carcinoma Cells

RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double-stranded RNA (dsRNA), also known as small activating RNA (saRNA). p21(WAF1/CIP1) (p21) is a putative tumor suppressor gene due to its role as a key negative regulator of the cell cycle and cell proliferation. It is frequently downregulated in cancer including hepatocellular carcinoma (HCC), but is rarely mutated or deleted, making it an ideal target for RNAa-based overexpression to restore its tumor suppressor function. In the present study, we investigated the antigrowth effects of p21 RNAa in HCC cells. Transfection of a p21 saRNA (dsP21-322) into HepG2 and Hep3B cells significantly induced the expression of p21 at both the mRNA and protein levels, and inhibited cell proliferation and survival. Further analysis of dsP21-322 transfected cells revealed that dsP21-322 arrested the cell cycle at the G(0)/G(1) phase in HepG2 cells but at G(2)/M phase in Hep3B cells which lack functional p53 and Rb genes, and induced both early and late stage apoptosis by activating caspase 3 in both cell lines. These results demonstrated that RNAa of p21 has in vitro antigrowth effects on HCC cells via impeding cell cycle progression and inducing apoptotic cell death. This study suggests that targeted activation of p21 by RNAa may be explored as a novel therapy for the treatment of HCC.     

Cyr61/CCN1 is a tumor suppressor in human hepatocellular carcinoma and involved in DNA damage response

Cyr61/CCN1 is a secreted extracellular matrix associated protein involved in diverse biological functions and plays multiple roles in tumorigenesis. Cyr61 was down-regulated in HCC tumor tissues as observed in our previous cDNA microarray study, but its potential role in hepatocarcinogenesis is still unclear. To explore the biological significance of Cyr61 in HCC development, over-expression of this gene was established in HCC cell lines and its effects on cell proliferation, adhesion, migration and invasion were analyzed in this study. Cyr61 expression was down-regulated in HCC tumors as measured by quantitative real-time PCR and its protein level was decreased in most HCC cell lines as detected by Western blot. Over-expression of Cyr61 in HCC cell lines suppressed cell proliferation in monolayer and anchorage-independent growth in soft agar, whereas down-regulation of Cyr61 by siRNA increased cell proliferation rate. Over-expression of Cyr61 also significantly enhanced adhesion activities of HepG2 cells to various ECM proteins. Moreover, stably transfected HepG2-Cyr61 cells showed inhibited cell mobility (40-45%) and reduced invasiveness (30-40%) compared to HepG2-Neo controls. Furthermore, upon exposure to 5-Fluorouracil and UV irradiation, Cyr61 was rapidly induced in both p53(+/+) HepG2 and p53(-/-) Hep3B cells. However, only HepG2 cells showed increased G2/M phase arrest with concomitant up-regulation in p53 and p21 levels, suggesting that Cyr61 may play an active role in regulating HCC cell growth involving p53 as well as alternative pathways. In conclusion, we demonstrated that Cyr61 is a tumor suppressor in hepatocarcinogenesis and is involved in DNA damage response.     

Up-regulation of cyclin D1 by HBx is mediated by NF-kappaB2/BCL3 complex through kappaB site of cyclin D1 promoter

Cyclin D1 is frequently overexpressed in hepatocellular carcinoma (HCC) exhibiting increased malignant phenotypes. It has also been known that the hepatitis Bx (HBx) protein is strongly associated with HCC development and progression. Although overexpression of both proteins is related to HCC, the relationship between the two has not been well studied. Here we show that HBx up-regulates cyclin D1 and that this process is mediated by the NF-kappaB2(p52)/BCL-3 complex. Our experiments indicate that HBx up-regulates BCL-3 in the mRNA level, which subsequently results in the up-regulation of the NF-kappaB2(p52)/BCL-3 complex in the nucleus. Moreover, impaired HBx-mediated BCL-3 up-regulation by small interfering RNA for BCL-3 reduced HBx-mediated cyclin D1 up-regulation. Down-regulation of the HBx protein level by p53 also reduced HBx-mediated cyclin D1 up-regulation. From these results, we conclude that the up-regulation of cyclin D1 by HBx is mediated by the up-regulation of NF-kappaB2(p52)/BCL-3 in the nucleus. This HBx-mediated-cyclin D1 up-regulation might play an important role in the HBx-mediated HCC development and progression.     

p21 promotes ceramide-induced apoptosis and antagonizes the antideath effect of Bcl-2 in human hepatocarcinoma cells

p21, a potent cyclin-dependent kinase inhibitor, has been known to induce cell cycle arrest in response to DNA-damaging agents. Although p21 has been reported to play an important role in the regulation of apoptosis, the postulated role for p21 in apoptosis is still controversial. Previously, we reported that p21 was induced in a p53-independent manner during ceramide-induced apoptosis in human hepatocarcinoma cell lines. In the present study, we investigated the precise role of p21 in ceramide-induced apoptosis in human hepatocarcinoma cells by using a tetracycline-inducible expression system. Overexpression of p21 by itself did not induce apoptosis in p53-deficient Hep3B cells. However, Hep3B/p21 cells were more sensitive to ceramide-induced apoptosis. In these cells, p21 overexpression did not result in G1 arrest. The expression level of Bax was increased in Hep3B/p21 cells treated with ceramide and its expression was more accelerated under the p21-overexpressed condition compared to that of the p21-repressed condition. Overexpression of Bax induced apoptosis in Hep3B cells. On the other hand, the levels of p21 and Bax protein were increased by ceramide in another hepatocarcinoma cell line, SK-Hep-1, while the Bcl-2 protein level was not changed. Overexpression of Bcl-2 not only suppressed apoptosis but also completely prevented induction of p21 and Bax caused by ceramide in SK-Hep-1 cells. Furthermore, overexpression of p21 antagonized the death-protective function of Bcl-2 and upregulated expression of Bax protein. These results suggest that p21 promotes ceramide-induced apoptosis by enhancing the expression of Bax, thereby modulating the molecular ratio of Bcl-2:Bax in human hepatocarcinoma cells.